1. Technical Field
The subject invention relates to methods of processing and producing recombinant proteins in vivo, for example, recombinant phosphorylated human beta-casein. The subject invention also relates to the proteins produced by these methods as well as to uses of these proteins.
2. Background Information
It is generally recognized that human milk is the best nutritional source for human infants. Human milk is not only an ideal source of nutrients for the developing infant, but it also contains immunoglobulins as well as non-immunological factors that protect the infant from infection by various microorganisms. Human milk is also easily digestable and is less likely to cause allergic reactions than infant formulae based on bovine milk.
Human milk differs from bovine milk, as well as the milk of other mammalian species, in various ways. In particular, overall protein content differs between human milk and bovine milk. Furthermore, bovine milk contains five caseins (i.e., 2 alpha-caseins, 1 beta-casein, 1 kappa-casein and 1 gamma-casein). In contrast, human milk contains only beta- and kappa-casein. Additionally, the amino acid sequences of human milk proteins differ from that of other mammalian milk proteins.
Efforts have been made to develop infant milk formulae that have some of the advantageous properties of human milk, yet which avoid the disadvantages associated with bovine milk based infant formulae such as, for example, allergic reactions and incomplete digestion by the infant. A desirable method to achieve this goal is to add some of the known constituents of human milk, including human milk proteins in their native form, to infant formulae. The human caseins represent important substances, which if added in their native form to infant formulae, would serve to enhance the nutritional value of the formulae and reduce the inherent disadvantages of non-human milk proteins. Human milk casein has also been credited with enhancing calcium absorption, inhibiting angiotensin I-converting enzyme, being an opioid agonist, and exhibiting immunostimulating and immunomodulating effects.
Furthermore, in addition to being a source of amino acids necessary for the synthesis of proteins required for the growth and development of infants, human milk is recognized as containing proteins, including casein, that have other important biological functions. Beta-casein, mentioned above, for example, is one of the most abundant milk proteins synthesized in the mammary gland. After post-translational modification in the Golgi apparatus, the protein is secreted as large calcium-dependent aggregates called micelles. Beta-casein is not a single entity. Rather, it is a heterogeneous group of phosphoproteins secreted during lactation in response to lactogenic hormones.
The primary structure of human beta-casein was determined by Greenberg et al. (Journal of Biological Chemistry 259:5132-38 (1984)). Human casein consists largely (&gt;80%) of the beta-form with a smaller amount in the kappa-form. Native beta-casein is a 25 kDa protein. In human milk, beta-casein molecules show variable degrees of post-translational phosphorylation ranging from zero to five phosphate groups per polypeptide chain (FIG. 1; Greenberg et al., supra (1984); Hansson et al., Protein Expression and Purification 4:373-81 (1993)). Phosphate groups in the native protein are attached to serine and threonine residues located near the amino terminus (Greenberg et al., supra (1984)). Human and bovine beta-casein exhibit 47% identity in their amino acid sequences.
In view of the benefits of beta-casein, it would be quite beneficial, as noted above, to add this protein to infant formulae or other nutritional formulae. Thus, methods must be devised to create and express this protein and, in particular, beta-casein, recombinantly. Yet, in the expression of proteins in bacterial systems, proteins are obtained which may be lengthened at the N-terminus by a methionine residue.
In a significant fraction of mature cytosolic proteins in bacteria, the N-terminal methionine is cleaved off by a methionine aminopeptidase whose specificity is dependent on the identity of the adjacent amino acid residue (Ben-Bassat et al., Journal of Bacteriology 169:751-57 (1987)). In the case of recombinant human beta-casein production in E. coli, the extra N-terminal methionine is not cleaved from the adjacent arginine residue by this peptidase (Hansson et al., supra (1993)). Since the methionine can be oxidized to sulfonyl methionine during purification, and this event can increase the immunogenicity/allergenicity of the protein, removal of this terminal methionine is desirable.
One method of generating recombinant protein with a desired N-terminal residue is to express the protein fused to a signal peptide at the N-terminus. This method leads to export, cleavage of the signal peptide, and accumulation of the processed, native protein in the periplasmic space of gram-negative bacteria or in the cytosol of gram-positive bacteria. The protein may be secreted and/or accumulated in the cytosol in yeast, fungi or mammalian cells. However, specificity of the peptidase cleaving the signal peptide may result in heterogeneity at the N-terminus (Lingappa et al., Proceeding of the National Academy of Science USA, 74:2432-36 (1977); Hirtzman et al., Science 219:620-25 (1983)). Also, there can be a significant quantity of residual cytosolic protein in which the signal peptide is not cleaved.
Another method of removing N-terminal methionine from a recombinant protein is to use purified aminopeptidases (e.g., methionine aminopeptidase, aminopeptidase M, dipeptidyl aminopeptidase) for in vitro processing of the purified recombinant protein (reviewed by Ben-Bassat in Purification and Analysis of Recombinant Proteins. eds. R. Seetharam and S. K. Sharma, pp.148-59, Marcel Dekker Inc., N.Y. (1991)). However, this method requires multiple purification steps and may not be economical for large-scale production.
Also, a process to produce proteins which begin at the N-terminus with proline has been described in German Patent Application P 38 11 921.8. This process involves enzymatic cleavage with aminopeptidase-P. Additionally, proline iminopeptidase has also been used in order to liberate a desired protein as described in Australian Patent Application AU-A-37170/89. Both of these processes are quite distinct from that described in the present invention. In fact, the present invention overcomes many disadvantages of these two methods, the methods described above, as well as all such methods utilized in the past.